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Membrane Protein Insertion. Dr. Ross Dalbey [email protected] Feb 3, 2012. Membrane Proteins. Significance: 25% of proteins in human cells are membrane proteins; membrane proteins make up more than 50% of the known drug targets. Membrane proteins play essential cell functions. - PowerPoint PPT Presentation
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Membrane ProteinsSignificance: 25% of proteins in human cells are membrane proteins; membrane proteins make up more than 50% of the known drug targets
ATPaseF1Fo
reactioncenter
H+
chemoreceptorTar translocase
SecYEG
lactosepermease
LacY
• energy conversion ATPase• energy gain photosynthetic complex• signalling chemosensors• substrate transport permeases• protein translocation translocases
Membrane proteins play essential cell functions
h.
Key Question: How do proteins insert into the membrane?
Current Understanding of Membrane Protein Insertion
Different Insertion Pathways: Spontaneous, SRP, Sec Translocase
The Discovery of YidC
YidC mediates membrane protein insertion in bacteriaJames C. Samuelson1, Minyong Chen1, Fenglei Jiang1, Ines Möller2, Martin Wiedmann2, Andreas Kuhn3, Gregory J. Phillips4 & Ross E. Dalbey1
Significance
• Our objective as scientists is to advance the knowledge of membrane protein insertion in humans.
• Humans contain the essential mitochondrial
YidC homolog Oxa1• The essential bacterial YidC is a potential
antibacterial target
N C
periplasm
cytoplasm
innermembrane
+11 +2+7+1
+1 0
YidC
+5 +1
-2 0
Oxa1pN
intermembrane space
innermembrane
mitochondrialmatrix
-7
C
+15
What we know about YidC and its mitochondrial homology Oxa1p
E. coli has 4485 genes compared to ~ 25,000 in humans
E. coli is used as model system
How did we discover YidC is a membrane insertase?
a)In vivo: Made a YidC depletion strain and showed insertion was inhibited
b)In vitro: i) Crosslink YidC to a membrane protein during membrane insertion
ii) Reconstitute membrane protein insertion with lipid vesicles containing only YidC
Main Question
Does YidC catalyze membrane insertion?
In vivo
Construction of YidC depletion strain
YidC depletion studies
• Grow overnight E.coli culture with arabinose• Wash cells to remove residual arabinose• Back-dilute into LB containing arabinose or glucose• Grow until a significant growth defect is observed (2.5 – 3 hrs)• Switch to minimal media for protein labeling with 35S-methionine
Lysozyme Proteinase K
SpheroplastBacteria
Inserted-Digested by PK
Non-inserted-Resistant to PK
Inserted Protein Uninserted Protein
PK PKPeri
IM
Cyto
IM
CytoCyto
Peri
OM
IM
PK - + PK - +SDS-PAGEGel
Assay for Membrane Protein Insertion
Effect of YidC depletion on Membrane Insertion
In Vitro
Can YidC be crosslinked to an inserting membrane protein?
Photocrosslinking Approach
In vitro photocrosslinking studies
• Synthesize radioactive truncated membrane protein• Membrane protein remains bound to ribosome since lacks stop
codons• Generate insertion intermediate with inverted inner membrane
vesicles• Introduce photoprobe • Shining UV light results in photocrosslinking
YidC and Ffh (SRP) interact with Pf3 coat
In vitro
Reconstitute insertion of a membrane protein using purified YidCincorporated into lipid vesicles
Reconstitution of YidC into large unilamellar vesicles (LUVs) (Serek et al, 2004, EMBO J. 23, 294-301)
purified YidC E. coli lipids
extruder
proteo-LUVs
mix
200 nm
YidC
YidC substrate
determinants
YidC function: as a foldase or
insertase
Structure
The Border region and TM organization
Function in inserting
multispanning membrane
proteins
periplasm
L7
N6
N23I330
L340
Y370
L372
E415
K416
E445R447
K480
S482
Y465
K493
M495
F509P510
E536
L535
Defining the Aqueous/ Transmembrane border regions
Undergraduate Projects
Kelsey Kerton
Abdul Wasey
Spontaneous YidC only Sec/YidC
YidC determinants
?